Unit 7 - Regulation of the cell cycle by cyclin-dependent kinases

Question 1

what are the three major checkpoints of the cell cycle?

Answer 1

1. G1/S “start” commitment phase
   - trigger DNA replication machinery
2. G2/M “entry into mitosis”
   - trigger mitosis machinery
3. metaphase/anaphase “exiting mitosis”
   - trigger completion of mitosis and proceed to cytokinesis

Question 2

what is cyclin-dependent kinase? how does its activity change in interphase and mitosis?

Answer 2

a serine/threonine specific protein kinase w/o activity unless combined with matching cyclin regulatory subunit

• CDK activity increases suddenly in mitosis (due to slowly increasing concentrations of cyclin from interphase through mitosis)
• -after mitosis is done, levels of both CDK and cyclin decrease

Question 3

mitotic CDK activation

Answer 3

mitotic CDK + M-cyclin = inactive M-CDK

• inhibitory kinase Wee1 and activating kinase phosphorylate it to make it inactive M-CDK-P-P
• activating phosphatase Cdc25 removes the inhibitory Wee1 phosphate, leaving active-M-CDK-P
• active M-CDK-P is positive feedback to activate more Cdc25 phosphatases to activate more M-CDKs

Question 4

mitotic CDK inactivation

Answer 4

1. cyclin subunit is ubiquitylated by E3 ubiquitin ligase
   - targets cyclin subunit for proteolytic destruction by proteosome
2. activating phosphate is removed from kinase subunit, leaving inactive CDK

Question 5

what is APC?

Answer 5

anaphase promoting complex (E3 ubiquitin ligase that turns off mitotic CDK at 3rd checkpoint from metaphase to anaphase)
-helps triggers start of anaphase by turning off CDK activity, and by promoting Xm separation by activating protease separase

Question 6

securin and separase interaction and what do they do?

Answer 6

securin (inhibitory protein) is bound to inactive separase (proteolytic enzyme)

• active APC ubiquilates and degrades securin, activating separase
• active separase helps trigger transition from metaphase to anaphase

Question 7

what is the original trigger for metaphase to anaphase transition? how is this a checkpoint?

Answer 7

tension on all kinetochores, which inhibits kinase activity

• if one Xm fails to attach to both poles, the kinase remains active, and blocks progression to anaphase
• when all kinetochores are under tension, APC becomes active, and triggers separation of chromatids through proteolysis of cohesion complex

Question 8

how many CDKs are needed to regulate cell cycle progression?

Answer 8

mulitple; each CDK has a unique substrate specificity and a unique time during cell cycle when it’s active
-different cyclin/CDK combos are needed at different points in the cell cycle

Question 9

what CDK/cyclin combos are needed in G1–>S, progression through S, and entry into mitosis?

Answer 9

G1–>S: cyclin D + CDK4/6
through S: cyclin A/E + CDK 2
into mitosis: cyclin B + CDK 1

Question 10

concentrations of S VS M cyclin throughout cell cycle

Answer 10

S cyclin builds up in middle of G1 phase, and peak/plateau at start of S phase until slowly drops at start of M phase

M cyclin: starts increasing at beginning of G2, then peak/plateau at beginning of M phase, and steeply drops in middle of M phase

Question 11

how can CDKs be regulated?

Answer 11

• damaged DNA (G1/S/G2)
• unfavorable extracellular environment (G1)
• incompletely replicated DNA (S/G2)
• Xm improperly attached to mitotic spindle (M)

Question 12

how can DNA damage prevent mitosis?

Answer 12

DNA damage activates protein kinases that phosphorylate p53 to stable and active form

• active p53 binds to regulatory region of p21 gene
• -if binds too long, causes apoptosis
• -if can manage to transcript/translate p21 mRNA, makes p21 (CDK inhibitor protein)
• –p21 binds the active G1/S-CDK and S-CDKs, thus inactivating them, and blocking entry into S phase until DNA is repaired

Question 13

breast cancer and DNA damage repair

Answer 13

BRCA 1/2 proteins are important in dsDNA break repair

• if either one is completely absent, cell is unable to repair dsDNA breaks, so mutation rates increase
• most cells with DNA damage (especially if very extensive) don’t progress through cell cycle, since prolonged checkpoint activation leads to apoptosis

Question 14

what is PARP? what does inhibiting it do?

Answer 14

poly(ADP)ribose polymerase

• pathway is important for ssDNA break repair
• if inhibited in BRCA-mutant cancer cells, the rate of apoptosis can be increased

Question 15

what is Cisplatin and what does it do?

Answer 15

cancer drug that induces dsDNA damage that cannot be repaired, and is so extensive that the cell undergoes apoptosis

Question 16

external controls of cell cycle?

Answer 16

work through modulation of cell growth

• needed to pass Start checkpoint in G1
• directly activate mitogens
• growth factors increase rate of PRO synthesis and reduce rate of PRO degredation, so cells can reach critical growth status for entry into cell cycle

Question 17

what do mitogens do?

Answer 17

directly stimulate progression from G0 into cell cycle

-include ligands of receptor tyrosine kinases that activate the MAPK pathway

Question 18

what does activation of MAPK pathway cause?

Answer 18

activation from ligands of tyrosine kinase causes transcription of genes that encode G1 CDK subunits
-this inactivates retinoblastoma PRO (Rb) and trigger synthesis of genes needed to progress into S phase